野生金荞麦高黄酮含量愈伤组织中一个类异黄酮还原酶(IRL)基因的克隆与分析(英文)

Molecular cloning and characterization of a novel isoflavone reductase-like gene(FcIRL) from high flavonoids-producing callus of Fagopyrum cymosum

木脂素是重要的植物防御物质之一,具有优良的生物学活性。为研究传统抗肿瘤良药野生金荞麦的药用次生代谢,用RACE与文库结合的方法,从野生金荞麦高黄酮含量愈伤系cDNA文库中快速克隆了一个类异黄酮还原酶(isoflavone reductase-like,IRL)基因FcIRL的全长cDNA(accession no.EU116032)。FcIRL cDNA全长1 217 bp,含有1个942 bp的开放阅读框(open reading frame,ORF),编码313个氨基酸,其5′UTR区含有2个终止子TAG,3′UTR区含有推测的加尾信号ATAAA和polyA尾,对应基因组序列不含内含子。FcIRL蛋白N端含有1个N-乙酰化作用位点(M1-K5)和1个保守的NADPH结合位点(G10-G-T-G13-Y-I-G16),具有1个保守的NmrA结构域(V6-N244)和多个重要的磷酸化位点,以及1个保守的N-糖基化作用位点(N214)。序列同源性比较、系统发生分析以及蛋白质高级结构预测分析均表明,FcIRL属于松脂醇-落叶松脂醇还原酶(pinoresinol-lariciresinol reductase,PLR)类,推测具有PLR的生物活性,是8-8′连接木脂素(8-8′-linked lignans)合成途径中的关键酶,催化松脂醇和落叶松脂醇,还原生成开环异落叶松脂(secoisolariciresinol),参与金荞麦药用次生代谢与植物的防御。

Lignans are important defensive compounds in plants and have good biological activities protecting human health. In order to study the medicinal secondary metabolism of Fagopyrum cymosum （Trev.） Meisn, a traditional Chinese medicine with anti-tumor effect, a novel isoflavone reductase-like gene, FcIRL, was cloned using RACE strategy from a cDNA library of high flavonoids-producing callus. The full-length cDNA of the FclRL was 1 217 bp （accession no. EUl16032）, which contained a 942 bp open reading frame （ORF） encoding a 313 amino acid protein. Two stop codons （TAG） and a putative polyadenylation signal ATAAA at 24 bp upstream from the polyadenylation site was found in 5＇ and 3＇ UTR, separately. And no intron was found in the genomic sequence yet. FcIRL contained a predicted N-terminal acetylation site （MrKs） and a NADPH-binding motif （G10-G-T-G13-Y-I-G16） in the N-terminal region, a conserved NmrA （nitrogen metabolite repression regulator） domain （V6-N244）, multi-phosphorylation sites and one conserved N-glycosylation site （N214）. Sequence homology comparison, phylogenetic analysis and advanced structures prediction all suggested that FcIRL belonged to the class of pinoresinol-lariciresinol reductase （PLR）, which is a key enzyme in synthetic pathway of 8-8＇-linked lignans, with function in catalyzing reduction of pinoresinol and lariciresinol into secoisolariciresinol, and medicinal secondary metabolism and resistance in F. cymosum.